HIV-1 latency is a major obstacle to effective, lifelong control of HIV-1 infection and has been the nemesis of curative strategies for the disease. Remarkably, estimates of the total body burden of latently-infected cells are in the range of only 106, a notably small number that would seem to represent a tractable target, nonetheless, previous attempts to eliminate these latently-infected cells using anti-CD3 monoclonal antibodies or Interleukin 2 (II-2)) have been unsuccessful. In this application, we propose a joint research effort between the University of Alabama at Birmingham and the Southern Research Institute (SRI) focusing on the discovery and validation of novel small molecule drug candidates that selectively activate HIV-1 expression in lymphoid cells. The proposed research builds on a body of previous work by SRI investigators and by our group (J Virol. 76:8776, 2002) that both demonstrates feasibility of the project and provides the critical cell-based assays needed for immediate ramp-up to high throughput screening (HTS) of compound libraries. Specific aims are: (i) to transfer an existing latently HIV-1 infected reporter cell line that uses enhanced green fluorescent protein as a quantitative marker of HIV-1 expression and is amenable to HTS in a 384-well plate format to the fully automated HTS platform at the SRI; (ii) to perform assay validation and initial screens of >1,000 compounds for activity in inducing HIV-1 expression; (iii) to generate second generation HIV-1 latency assays that will minimize false negative or positive hits; (iv) to develop protocols that allow for the evaluation of the ability of lead compounds identified by HTS to induce HIV-1 expression in virally-infected CD4+ memory T lymphocytes from HIV-infected patients. The proposed research capitalizes on unique and proven strengths at DAB and SRI in molecular virology and drug discovery and promises to provide a scientific and technical platform that will allow for the future screening of large compound libraries with the objective to discover and preclinically develop HIV-1 reactivating agents. Integration of HIV-1 reactivating agents into the existing HIV-1 treatment schedule with the goal to deplete the cellular reservoirs of latent HIV-1 will be a first essential step towards the development of a therapeutic treatment strategy for HIV-1 infection.